Activity-based probe compounds, compositions, and methods of use thereof

Abstract

The present invention provides a probe compound for labeling a cysteine protease based on activity. The compound targets the protease through a specific targeting element. The compound also includes a detectable element, such as a fluorescent label, a radioactive label, or a chelator. In some cases, the compound also includes a quenching element that is released upon reaction with the protease. The present invention also provides compositions comprising the compound, as well as methods of using the compound, such as labeling a protease in an animal and visualizing a tumor in an animal.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 438,959, filed December 23, 2016, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Government Support Statement

[0004] This invention was developed under a government-supported contract EB005011 (issued by the National Institutes of Health). The government holds specific rights to this invention. Background Technology

[0005] Various technologies are currently being developed for use in the fields of molecular imaging and disease monitoring. In particular, optical fluorescence imaging is a method that is beginning to show potential as a clinical tool due to its sensitivity, specificity, and non-invasiveness. The specificity of fluorescent optical probes can be provided in some cases by their biological targets. For example, if the fluorescence of a probe is released only in the presence of an enzyme reaction, the identification of other optical probes by enzyme targets in biological samples often produces a very specific signal. Ideally, even after the fluorescent signal has been activated by an enzyme reaction, the fluorescent portion of the probe remains linked to its enzyme target. The use of such fluorescently active probes (ABPs) for protease targets has been disclosed. Blum et al., (2009) PLoS One 4:e6374; doi:10.1371 / journal.pone.0006374. ABPs can be distinguished from simple fluorescent substrates by the permanent covalent bond formed by the reaction between the ABP and the catalytic residues of the enzyme's active site. Although fluorescent substrates may seem advantageous due to signal amplification caused by catalytic reversal of the target enzyme, it has been found that APB exhibits increased tissue uptake kinetics and prolonged probe retention in target tissues due to covalent modification of the target enzyme.

[0006] Among the target enzymes of interest for use with fluorescence-based optical probes are proteases, particularly cysteine ​​proteases. Cysteine ​​cathepsins are a family of proteases that play important roles in health and disease. (Reiser et al., (2010) J. Clin. Invest. 120:3421-31). Although their functions have been primarily described as being limited to the endosome pathway, mounting evidence suggests they are major regulators of matrix degradation, indicating that they also play a role extracellularly. &Wilson (2011) Role of Cysteine ​​Cathepsins in Extracellular Proteolysis. Biology of Extracellular Matrix Volume 2 23-51. Furthermore, members of the cysteine ​​cathepsin family have been shown to be key players in the development and progression of several types of cancer. Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75; Palermo & Joyce (2008) Trends Pharmacol. Sci. 29:22-8. In addition, altered expression of endogenous inhibitors of cysteine ​​cathepsins (cysteine ​​protease inhibitors) has been observed in cancer. Cox (2009) Cystatins and cancer. Front. Biosci. 14:463-74. These observations, combined with potential changes in the intracellular and extracellular environments, underscore the importance of tools that allow for the direct assessment of the activity of these proteases within the context of the native tumor microenvironment. Several ABPs targeting the cysteine ​​cathepsin family have been synthesized. Edgington et al., (2011) Curr. Opin. Chem. Biol. 15:798-805. In particular, fluorescence-quenched ABP (qABP) has proven to be a powerful tool for non-invasive optical imaging of cancer at the tissue, cellular, and protein levels, as well as for subsequent characterization of target cathepsins. Blum et al., (2007) Nat. Chem. Biol. 3:668-77; Verdoes et al., (2012) Chem. Biol. 19:619-28.

[0007] Inhibitors of active dipeptidase I based on the reactive group of 2,3,5,6-tetrafluorophenoxyaryl methyl ketone have been reported (Deu et al., (2010) Chem Biol. 17: 808-819), but these inhibitors are non-peptide and do not include detectable groups.

[0008] Activity-based peptide inhibitors for quenching in fluorescence imaging of cells containing active proteases, such as cathepsins, have also been reported. See, for example, U.S. Patent Application Publication No. 2007 / 0036725. These probes employ an ester-linked acyloxymethyl ketone reactive group to bind to the protease active site. In some cases, activity-based fluorescent probes are non-peptide probes. See, for example, PCT International Publication No. WO 2012 / 118715. In some cases, activity-based probes are used to radiolabel their target enzymes. See, for example, PCT International Publication No. WO 2009 / 124265.

[0009] Other activity-based caspase and other cysteine ​​protease inhibitors are reported in PCT International Publication No. WO 2012 / 021800; US Patent Application Publication No. 2002 / 0052323; US Patent Application Publication No. 2002 / 0028774; PCT International Publication No. WO 96 / 41638 and European Patent Application Publication No. EP0272671.

[0010] However, there is still a need in this field for novel fluorescent probes based on active cysteine ​​proteases that offer higher cellular uptake rates, target a broader spectrum of cysteine ​​protease activities, and provide improved detection sensitivity and lower background signal. Summary of the Invention

[0011] The present invention solves the above-mentioned and other problems by providing compounds, compositions for labeling cysteine ​​proteases, and methods of using said compounds and compositions.

[0012] In particular, according to one aspect of the invention, a compound represented by structural formula (II) is provided:

[0013]

[0014] Where D contains benzoindole dyes;

[0015] L1 is a linking group;

[0016] AA1 is an amino acid side chain;

[0017] U is O, NH, or S;

[0018] R1 is an alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl or protecting group, and is optionally substituted by one to three A groups;

[0019] Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, arylalkoxy, arylanoyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylanoyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloanoyl, cycloamino, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkylamino, hydroxyl, thiol, amino, alkanoylamino, arylanoylamino, alkylcarboxyl, carbonate, carbamate, guanidinyl, ureyl, halogen, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino, or azide;

[0020] L3 is a linking group; and

[0021] Q contains a quenching agent.

[0022] In some embodiments, the benzoindole dye has the following structure:

[0023]

[0024] Where o is an integer from 1 to 4;

[0025] R2 is a C2-C8 alkyl group, optionally substituted with a sulfonate or carbonate ester;

[0026] Each R3 is independently a C1-C6 alkyl group; and

[0027] L4 is an alkyl linking group with optional substitution, wherein each carbon atom is arbitrarily replaced by a heteroatom.

[0028] In a more specific embodiment, the benzoindole dye has the following structure:

[0029]

[0030] In embodiments of the compound of structural formula (II), L1 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced by a heteroatom, AA1 is an aralkyl amino acid side chain, optionally replaced by one to three A groups, U is O, L3 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced by a heteroatom, or L3-Q is

[0031]

[0032] Where R includes QSY quencher or QC-1 quencher; and

[0033] n is an integer from 1 to 8. More specifically, the QSY quencher can be a hydrophilic QSY quencher or a sulfonated QSY quencher. In some embodiments, the QC-1 quencher has the following structure:

[0034]

[0035] In other embodiments, the compounds of the present invention have formula (III):

[0036]

[0037] Where R includes either QSY quencher or QC-1 quencher; and M and n are independently integers from 1 to 8; and R1, AA1, and D are as defined above. More specifically, in these compounds, R can be...

[0038]

[0039] And D can be

[0040]

[0041] In even more specific embodiments, the compound may have the following structure:

[0042]

[0043] According to another aspect of the invention, the invention provides a composition comprising the compound of the invention and a pharmaceutically acceptable carrier for labeling proteases in animals.

[0044] According to another aspect of the present invention, the present invention provides a method for labeling proteases in an animal, the method comprising the following steps:

[0045] Administer the composition of the present invention to animals.

[0046] The present invention also provides a method for visualizing tumors in animals.

[0047] Includes the following steps:

[0048] The composition of the present invention was administered to an animal, and a detectable signal was measured in the animal due to the reaction between the composition and the cathepsin cysteine ​​protease, wherein the detectable signal was associated with a tumor in the animal.

[0049] In certain method implementations, the detectable signal is a fluorescent signal. In other specific method implementations, the fluorescent signal is generated at the tumor margin. Attached Figure Description

[0050] Figure 1A The structure of qABP GB137(1) and probes 2 to 8. Figure 1B Labeling patterns of probes 1 to 8 at 1 μM in live RAW cells. Figure 1C Concentration-dependent labeling of probes 1 and 8 in live RAW cells. Figure 1D The intensity of probe 1-8 labeled with total cathepsin relative to 5 μM GB137(1) in live RAW cells.

[0051] Figure 2A At pH 5.5, probe 8 was used to label RAW cell lysates in a concentration-dependent manner. Figure 2B : Labeling time course of 0.5 μM probe 8 in live RAW cells. Figure 2CIn live RAW cells, pretreatment with JPM-OEt (50 μM) was used to inhibit the labeling of probes 1 and 8 and to improve serum stability. Figure 2D Live-cell fluorescence micrographs of RAW cells exposed to 1 μM probe 8 (top row) or micrographs co-localized with lysosomal tracer (second row, scale bar: 10 μm).

[0052] Figure 3A Time-process diagram of non-invasive optical imaging of tumor-bearing mice injected with probes 8 and 1 (right figure). The figure below shows the best fluorescence contrast at each time point. Figure 3B Time-dependent tumor-specific fluorescence (tumor-background) in mice treated with probes 1 or 8 (n=3; data represent mean ± standard error). Figure 3C The image shows the fluorescence of the tumor in vitro (top) and the fluorescently labeled proteins in vivo (bottom) visualized by in-gel fluorescence scanning after SDS-PAGE. Figure 3D Fluorescence intensity at the endpoints of non-invasive optical imaging (shown in 3A), ex vivo tumor imaging, and intragel fluorescent labeling (shown in 3C). Intensities are shown relative to probe 1 (n=3; data represent mean ± standard error). Figure 3E Microscopic images of fluorescence in tumor tissue sections treated with probe 8 (left), showing CD68 immunostaining (middle) and nuclear staining (DAPI - right, scale bar: 50 μm). Figure 3F : 3D reconstruction of CLSM of tumor tissue sections treated with probe 8 (originally red), with CD68 immunostaining (originally green) and nuclear staining (DAPI – originally blue).

[0053] Figure 4A Immunoprecipitation of BMV109 (probe 8) labeled cysteine ​​cathepsins. Figure 4B and 4C In live RAW cells, probes 1-8 were labeled in a concentration-dependent manner. Groups 4B and 4C were run on the same gel.

[0054] Figure 5A Non-invasive optical imaging of tumor-bearing mice 8 hours after injection of probes 1, 2, 6, or 8. The figures below show the best fluorescence contrast at each time point. Figure 5B Time-dependent tumor-specific fluorescence (tumor-background) in mice treated with probes 1, 2, 6, or 8 (n = 3; data represent mean ± standard error). Figure 5C The image shows the fluorescence of the tumor in vitro (top) and the fluorescently labeled proteins in vivo (bottom) visualized by in-gel fluorescence scanning after SDS-PAGE. Figure 5DFluorescence intensity at the endpoints of noninvasive optical imaging (shown in 5A), ex vivo tumor imaging, and intragel fluorescent labeling (shown in 5C). Intensities are shown relative to probe 1 (n=3; data represent mean ± standard error). Figure 5E Fluorescence micrographs of tumor tissue sections treated with probe 8 (columns 1, 3, and 4), with CD68 immunostaining (columns 2, 3, and 4) and nuclear staining (DAPI, columns 3 and 4, scale bar: 50 μm). A probe-free control (middle row) and an atypical control with immunostaining are shown (bottom). Figure 5F Co-location diagram of probe 8 (Cy5) and CD68 (FITC).

[0055] Figure 6A Comparison of in vivo and in vitro data of BMV109-Dylight780 and BMV109-ICG (10 nmol, 24 hours, Pearl, ex / em = 785 / 820 nm). Figure 6B In vitro studies of BMV109-Dylight780 and BMV109-ICG at different concentrations (10 nmol, 50 nmol, 100 nmol, 24 hours, Pearl, ex / em = 785 / 820 nm). Detailed Implementation

[0056] Cysteine ​​cathepsins are a family of proteases that play important roles in normal cell physiology and in the pathology of many human diseases. Therefore, numerous substrate and activity-based probe (ABP) classes have been developed to study the function of these enzymes. This paper presents a class of probes based on quenched fluorescence activity, which in some embodiments comprise an electrophilic phenoxymethyl ketone (PMK). Compared to previously reported ABPs, these reagents exhibit enhanced broad-spectrum reactivity to cysteine ​​cathepsins, resulting in significantly improved labeling performance both in vitro and in vivo. Furthermore, this paper demonstrates that the probes display tumors with unprecedented signal intensity and contrast in mice. These novel reagents enable the study of cysteine ​​cathepsins at the organismal, tissue, cellular, and protein levels in a variety of human disease models. Examples of such reagents are described in PCT International Publication No. WO2014 / 145257, which is incorporated herein by reference in its entirety.

[0057] compound

[0058] Therefore, in some aspects, the present invention provides a novel compound for labeling proteases, particularly cathepsins. The compound of the present invention may be a compound of formula (I):

[0059]

[0060] in

[0061] L is the leaving element connected to the ether;

[0062] T is the target element; and

[0063] D is a detectable element.

[0064] The target element T of the compound of the present invention can be a peptide or a non-peptide structure, and preferably the compound targets cysteine ​​protease.

[0065] Non-limiting examples of non-peptide structural elements incorporated for these purposes into the compounds of the present invention are those described in PCT International Publication No. WO2012 / 118715, the entire contents of which are incorporated herein by reference. In a preferred embodiment, the non-peptide target element comprises a triazole structure.

[0066] Specific examples of the compounds of the present invention having non-peptide target elements are:

[0067]

[0068] Non-limiting examples of peptide structural elements that can be used to target compounds to cysteine ​​proteases (especially cysteine ​​cathepsins) and can be incorporated into compounds of the present invention are those described in PCT International Publication No. WO2009 / 124265, the entire contents of which are incorporated herein by reference.

[0069] In some embodiments of the compounds of the present invention, DT- is...

[0070]

[0071] Where L1 is a linking group;

[0072] AA1 is an amino acid side chain;

[0073] U is O, N, or S;

[0074] R1 is an alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl, or protecting group, and optionally substituted with one to three A groups; and

[0075] Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, arylalkoxy, arylalkylacyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylalkylacyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkylamino, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkylamino, hydroxyl, thiol, amino, alkanoylamino, arylalkylamino, alkylcarboxyl, carbonate, carbamate, guanidinyl, urea, halogen, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino, or azide.

[0076] As used herein, the term "alkyl" refers to a saturated aliphatic group, including straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl. In some embodiments, the straight-chain or branched alkyl group has fewer than 30 carbon atoms in its backbone (e.g., straight-chain C1-C1). 30 C3-C branch 30 More specifically, fewer than 20. Similarly, some cycloalkyl groups have 3 to 10 carbon atoms in their ring structure, more specifically 5, 6 or 7 carbon atoms in their ring structure.

[0077] Furthermore, as used throughout the specification, examples, and claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl," where a substituted alkyl refers to an alkyl moiety having a substituent that replaces a hydrogen atom on one or more carbons of the hydrocarbon backbone. Such substituents may, for example, include halogen groups, hydroxyl groups, carbonyl groups (e.g., ketones, carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyl groups (e.g., thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate esters, phosphonates, hypophosphinates, amino groups, amide groups, amido groups, imino groups, cyano groups, nitro groups, azide groups, thioyl groups, alkylthio groups, sulfate esters, sulfonates, aminosulfonyl groups, sulfonyl groups, heterocyclic groups, aralkyl groups, aromatic or heteroaromatic moieties. Those skilled in the art will understand that the substituted portion on the hydrocarbon chain can itself be substituted if desired. For example, substituents in substituted alkyl groups may include substituted and unsubstituted forms of amino, azide, imino, amide, phosphoryl (including phosphonates and hypophosphonates) and sulfonyl (including sulfates, sulfonamides, aminosulfonyls, and sulfonates) and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylic acids, and esters), -CF3, -CN, etc. Exemplary substituted alkyl groups are described below. Cycloalkyl groups may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl groups, -CF3, -CN, etc.

[0078] In some specific embodiments, the term "alkoxy" as used herein refers to a lower alkyl group in which an oxygen atom is attached. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, n-butoxy, etc.

[0079] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to a substituent on one or more carbons of the alkenyl group that have substituted hydrogens. Such substituents may occur on one or more carbons, including or not including them in one or more double bonds. Furthermore, unless stability is inhibited, such substituents include all those intended for use with alkyl groups as discussed above. For example, substitution of the alkenyl group with one or more alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups is to be expected.

[0080] When used in conjunction with a chemical group (such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy), the term "C" is used. x-y "This aims to include groups containing x to y carbon atoms in the chain. For example, the term "C..." x-y "" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched alkyl groups containing x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. "C0-alkyl" indicates that the terminal group is hydrogen or, if internal, a bond. The term "C"... 2-y -olefin" and "C 2-y "-Alkyne" refers to substituted or unsubstituted unsaturated aliphatic group analogues that are similar to the alkyl groups described above in length and possible degree of substitution, but each contains at least one double or triple bond.

[0081] As used herein, the term "alkylamino" refers to an amino group that is substituted with at least one alkyl group.

[0082] As used herein, the term "alkylthio" refers to a thio group substituted with an alkyl group and can be represented by the general formula alkyl-S-.

[0083] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter referring to a substituent on one or more carbons of the alkynyl group that has substituted hydrogen. Such substituents may occur on one or more carbons, including or not including in one or more triple bonds. Furthermore, unless stability is inhibited, such substituents include all those substituents intended for use with alkyl groups as discussed above. For example, substitution of the alkynyl group with one or more alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups is to be expected.

[0084] As used in this article, the term "amide" refers to the following groups:

[0085]

[0086] Where R x and R y Each independently represents a hydrogen or hydrocarbon group or R x and R y Together with the N atoms they are attached to, they form heterocyclic rings with ring structures of 4 to 8 atoms.

[0087] The terms "amine" and "amino" are well known in the art and refer to unsubstituted and substituted amines and their salts, such as groups represented by the following formula:

[0088]

[0089] Where R x R y and R z Each independently represents a hydrogen or hydrocarbon group or R x and R y Together with the N atoms they are attached to, they form heterocyclic rings with ring structures of 4 to 8 atoms.

[0090] As used in this article, the term "aminoalkyl" refers to an alkyl group that has been substituted with an amino group.

[0091] As used herein, the term "aralkyl" refers to an alkyl group that has been substituted with an aryl group.

[0092] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is a carbon atom. In some embodiments, the ring is a 5- to 7-membered ring, and in more specific embodiments, a 6-membered ring. The term "aryl" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared in two adjacent rings, wherein at least one of the adjacent rings is aromatic; for example, the other rings may be cycloalkyl, cycloalkenyl, cycloynyl, aryl, heteroaryl, and / or heterocyclic. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0093] The term "carbamate group" is well known in the art and refers to the following group:

[0094]

[0095] Where R x and R y Independently representing hydrogen or hydrocarbon groups or R x and R y Together with the atoms they are attached to, they form heterocyclic rings with ring structures of 4 to 8 atoms.

[0096] As used herein, the term "cycloalkyl" refers to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. In some embodiments, the cycloalkyl ring comprises 3 to 10 atoms, and in more specific embodiments, 5 to 7 atoms.

[0097] The term "carbonate group" is well known in the art and refers to the group -OCO2-R. x , where R x It indicates a hydrocarbon group.

[0098] As used in this article, the term "carboxyl group" refers to a group represented by the formula -CO2H.

[0099] As used in this article, the term "ester" refers to the group -C(O)OR. x , where R x It indicates a hydrocarbon group.

[0100] As used herein, the term "ether" refers to a hydrocarbon group that is attached to another hydrocarbon group via oxygen. Therefore, the ether substituent of a hydrocarbon group can be alkyl-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocyclic-O-heterocycles and aryl-O-heterocycles. Ethers include an "alkoxyalkyl" group, which can be represented by the general formula alkyl-O-alkyl.

[0101] The term "guanidinyl" is well known in the art and can be represented by the following general formula:

[0102]

[0103] Where R x and R y Independently represents either hydrogen or hydrocarbon groups.

[0104] As used in this article, the terms “halogenated” and “halogen” refer to halogens and include fluorine, chlorine, bromine and iodine.

[0105] As used herein, the terms “hetaralkyl” and “heteroaralkyl” refer to alkyl groups having heteroaryl substitutions.

[0106] The terms "heteroaryl" and "hetaryl" encompass substituted or unsubstituted aromatic monocyclic structures, in some embodiments 5- to 7-membered rings, more specifically 5- to 6-membered rings, whose ring structure includes at least one heteroatom, in some embodiments 1 to 4 heteroatoms, and in more specific embodiments one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic systems having two or more rings, wherein two or more carbons share two adjacent rings, wherein at least one of the rings is heteroaromatic; for example, the other rings may be cycloalkyl, cycloalkenyl, cycloynyl, aryl, heteroaryl, and / or heterocyclic. Heteroaryl groups (e.g.) include pyrrole, furan, thiophene, imidazole, etc. Zyrazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, etc.

[0107] As used in this article, "heteroatom" refers to an atom of any element other than carbon or hydrogen. Typical heteroatoms are nitrogen, oxygen, and sulfur.

[0108] The terms "heterocyclic group," "heterocyclic," and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, in some specific embodiments a 3- to 10-membered ring, more specifically a 3- to 7-membered ring, whose ring structure includes at least one heteroatom, in some embodiments one to four heteroatoms, and in more specific embodiments one or two heteroatoms. The terms "heterocyclic group" and "heterocyclic" also include polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one of said rings is a heterocyclic group, and the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Heterocyclic groups (e.g.) include piperidine, piperazine, pyrrolidine, morpholine, lactone, and lactam, etc.

[0109] As used herein, the term "heterocyclic alkyl" refers to an alkyl group that has been substituted with a heterocyclic group.

[0110] As used herein, the term "hydrocarbon group" refers to a group bonded by carbon atoms that does not have a =O or =S substituent and typically has at least one carbon-hydrogen bond and a substantially carbon skeleton, but may optionally include heteroatoms. Therefore, for this purpose, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbon groups, but substituents such as acetyl (which has a =O substituent on the connecting carbon) and ethoxy (which is linked by oxygen rather than carbon) are not considered hydrocarbon groups. The hydrocarbon groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, ynyl, and combinations thereof.

[0111] As used in this article, the term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group.

[0112] When the term "lower" is used in conjunction with a chemical group (such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy), it refers to a group in which there are ten or fewer non-hydrogen atoms in the substituents, and in some embodiments, six or fewer. "Lower alkyl," for example, refers to an alkyl group containing ten or fewer carbon atoms, and in specific embodiments, six or fewer carbon atoms. In some embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, and alkoxy substituents as defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, and lower alkoxy, whether they appear alone or in combination with other substituents, as defined in hydroxyalkyl and aralkyl groups (in which case, for example, atoms in the aryl group are not counted when calculating carbon atoms in the alkyl substituents).

[0113] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic) where two or more atoms are shared between two adjacent rings; for example, the rings are "fused rings." The rings of a polycyclic compound can be substituted or unsubstituted. In some embodiments, the rings of a polycyclic compound are rings containing 3 to 10 atoms, more specifically 5 to 7 atoms.

[0114] The term "substituted" refers to a group comprising a substituent that replaces hydrogen atom on one or more carbon atoms in the backbone. It should be understood that "substituted" or "substituted" includes the implicit condition that such substitution is based on the permissible valence of the substituted atom and the substituent, and that the substitution produces a stable compound, for example, one that does not spontaneously transform under its conditions of use (e.g., through rearrangement, cyclization, elimination, etc.). As used herein, "substituted" means including all permissible substituents in organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds. For a suitable organic compound, permissible substituents can be one or more and can be the same or different. For the purposes of this invention, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any permissible substituents in organic compounds that satisfy the heteroatom valence as described herein. Substituents may include any substituents described herein, such as halogens, hydroxyl groups, carbonyl groups (e.g., ketones, carboxyl groups, alkoxycarbonyl groups, formyl groups, or acyl groups), thiocarbonyl groups (e.g., thioesters, thioacetic acid esters, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonates, hypophosphonates, amino groups, amide groups, amido groups, imino groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate groups, sulfonates, aminosulfonyl groups, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the substituted portion of the hydrocarbon chain may itself be substituted, if appropriate.

[0115] Unless otherwise specified, the chemical part “unsubstituted” as used herein shall be understood to include substituted variants. For example, reference to “aryl” group or part thereof implies both substituted and unsubstituted variants.

[0116] The term "sulfate" is well known in the art and refers to the group -OSO3H or its pharmaceutically acceptable salt.

[0117] The term "sulfonamide" is well known in the art and refers to a group represented by the following general formula:

[0118]

[0119] Where R x and R y Independently represents either hydrogen or hydrocarbon groups.

[0120] The term "sulfoxide" is well known in the art and refers to the group -S(O)-R. x , where R x It indicates a hydrocarbon group.

[0121] The terms “sulfonyl” or “sulfonate group” are well known in the art and refer to the group -SO3H or its pharmaceutically acceptable salt.

[0122] The term "sulfone" is well known in the art and refers to the group -S(O)2-R. x , where R x It indicates a hydrocarbon group.

[0123] As used in this article, the term "thioalkyl" refers to an alkyl group that has been substituted with a mercapto group.

[0124] As used in this article, the term "thioester" refers to the group -C(O)SR. x or -SC(O)R x , where R x It indicates a hydrocarbon group.

[0125] As used in this article, the term "thioether" is equivalent to an ether in which oxygen is replaced by sulfur.

[0126] The term "urea group" is well known in the art and can be represented by the following general formula:

[0127]

[0128] Where R x and R y Independently represents hydrogen or hydrocarbon groups.

[0129] The compounds of this invention are generally synthesized using standard synthetic chemistry techniques, for example, using the methods described in the Examples section below. Other useful synthetic techniques (e.g.) are described in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Ed. (Wiley, 2013); Carey and Sundberg, Advanced Organic Chemistry 4 thEd., Vols. A and B (Plenum 2000, 2001); Fiesers' Reagents for Organic Synthesis, Volumes 1-27 (Wiley, 2013); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-81 (Wiley, 2013); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989) (the entire contents of which are incorporated herein by reference). These compounds are typically synthesized using commercially available starting materials or can be readily prepared using methods well-known to those skilled in the art. For example, see Fiesers' Reagents for Organic Synthesis, Volumes 1-27 (Wiley, 2013) or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including supplements.

[0130] When referring to the components of the compounds of the present invention, the term "residue derived from..." may be used to describe residues formed by the reaction of a first reactive functional group on a first component and a second reactive functional group on a second component to form a covalent bond. In an exemplary embodiment, an amine group on the first component may react with an activated carboxyl group on the second component to form a residue comprising one or more amide moieties. Other arrangements of the first and second reactive functional groups are covered by the present invention. For example, as those skilled in the art will appreciate, a copper-catalyzed or copper-free reaction between an azide-substituted first component and an alkyne-substituted second component via a known "click" reaction produces a residue comprising a triazole. See Kolb et al., (2001) Angew. Chem. Int. Ed. Engl. 40:2004; Evans (2007) Aus. J. Chem. 60:384. An exemplary method for generating non-peptide fluorescent imaging probes using a "click" reaction is provided in PCT International Publication No. WO 2012 / 118715. Those skilled in the art can modify these methods to produce or modify the compounds claimed in this invention.

[0131] Those skilled in the art will understand that protecting groups are reversibly attached to desired positions on a molecule to control the reaction of other reagents at that position. Protecting groups useful in the practice of this invention are well known in the art. See, for example, Greene's Protective Groups in Organic Synthesis, edited by PGMWuts and TWGreene, 4. th edition (Wiley-Interscience, 2006); and Protecting Groups by P. Kocienski (Thieme, 2005).

[0132] The L1 group of the compound of the present invention is a linking group that connects the detectable element D to the target element. As will be understood by those skilled in the art, this group can be any suitable linking group. The L1 group is preferably an alkyl linking group, wherein the alkyl linking group is optionally substituted, and furthermore, the carbon in the linking group is optionally replaced by a heteroatom to achieve a chemically stable structure. Such substitution and replacement should be understood to include the insertion of groups such as ethers, thioethers, disulfides, esters, amides, carbonates, carbamates, etc., into the linking group. Preferably, the linking group is in the range of 5 to 40 bond lengths and can be branched, straight-chain, or contain rings. In some cases, the linking group may include double bonds. Depending on specific needs, they may be hydrophobic or hydrophilic as required.

[0133] It should also be understood that the connection between the L1 group and the detectable element D can be any suitable chemical connection, as will be understood by those skilled in the art. For example, in some cases, the compounds of the present invention may be readily prepared by including a portion in the precursor of the detectable element that reacts with a specific chemical group (e.g., amino, thiol, etc.). The detectable element can be readily attached to the target element at such a location by the reaction of this group on the target element. It is thus understood that these types of connections are within the scope of the compounds disclosed herein, even if the details of the connection structure are not explicitly shown.

[0134] As will be understood by those skilled in the art, the AA1 group of the compounds of the present invention can be any natural or non-natural amino acid side chain. In a preferred embodiment, the AA1 group is an aralkyl amino acid side chain optionally substituted with 1 to 3 A groups. In an even more preferred embodiment, the AA1 group is a phenylalanine side chain.

[0135] In the preferred compound, the U group is O.

[0136] In certain embodiments, the detectable elements of the compounds of the present invention are fluorescent labels, radioactive labels, chelating agents, etc. Examples of radioactive labels and chelating agents suitable for use in these compounds are described in PCT International Publication No. 2009 / 124265.

[0137] In a preferred embodiment of the compound of the present invention, the detectable element is a fluorescent label. As is known to those skilled in the art, when stimulated by the absorption of incident electromagnetic radiation, the fluorescent label emits electromagnetic radiation, preferably visible light. A wide variety of commercially available fluorescent labels are available, including labels with reactive moieties for coupling the label to a reactive group, such as an amino group, a thiol group, etc. See, for example, *The Molecular*. The entire contents of the Handbook—A Guide to Fluorescent Probes and Labeling Technologies are incorporated herein by reference.

[0138] An example of fluorescent labeling is fluorescein, which is widely used for immunofluorescence labeling. Fluorescein is a succinyl fluorescein dye with maximum absorbance at 495 nm. The associated fluorophore is Oregon Green, a fluorinated derivative of fluorescein.

[0139] In some embodiments, the fluorescent label used in the detectable element of the compounds of the present invention may be a pH-dependent fluorophore. As will be understood by those skilled in the art, such fluorescent labels, for example, as used in compounds labeled “LES12” and “LES13” as shown below, exhibit a fluorescence spectrum that depends on the pH of the labeling environment and can therefore be used to report environmental information about the labeling after the reaction, such as information about the location or type of the protease labeled by the reacting compound. Various useful pH-dependent fluorophores of different labels included in the detectable element of the compounds of the present invention are known, see, for example, The Molecular Handbook—A Guide to Fluorescent Probes and Labeling Technologies.

[0140] Other exemplary fluorescent labels suitable for use in the compounds of this invention are fluoroboron fluorescent dyes, rhodamine, and anthocyanin dyes. In particular, fluoroboron fluorescent dyes are represented by 4,4-difluoro-4-borona3a,4a-diaza-s-benzodiindene (boron fluoride complexed with dipyrrolemethyl methacrylate), and are referred to as... Dyes. Various derivatives of these dyes are known and considered suitable for use as detectable elements in the compounds of the present invention. See, for example, Chen et al., (2000) J.Org.Chem.65:2900-2906.

[0141] Another useful class of fluorescent labels used in the compounds of this invention are IRDye infrared dyes available from Li-Cor (www.licor.com). Non-limiting examples of these dyes are IRDye 800CW, IRDye 680RD, IRDye 680LT, IRDye 750, IRDye 700DX, IRDye 800RS, and IRDye 650.

[0142] Rhodamine dyes are a class of dyes based on the rhodamine ring structure. Rhodamine specifically includes tetramethylrhodamine (TMR) and carboxytetramethylrhodamine (TAMRA). TMR is a very common fluorophore used in the preparation of protein conjugates (especially antibody and avidin conjugates), while TAMRA is a dye commonly used for oligonucleotide labeling and automated nucleic acid sequencing. Rhodamine exists as a natural complement to fluorescein-based fluorophores, providing longer wavelength emission maxima and thus offering opportunities for multicolor labeling or staining.

[0143] The sulfonated rhodamine series of fluorophores, known as Alexa Fluor dyes, are also included in the group of rhodamine dyes. Introduced by Molecular Probes, Alexa Fluor dyes exemplify a significant advancement in modern fluorescence techniques. These sulfonated rhodamine derivatives exhibit higher quantum yields than spectrally similar probes for more intense fluorescence emission and possess several additional improved properties, including enhanced photostability, absorption spectra matching common laser lines, pH insensitivity, and high water solubility.

[0144] Cyanide dyes correspond to the related dye families Cy2, Cy3, Cy5, Cy7, and their derivatives. They are based on a partially saturated indole nitrogen heterocyclic core with two aromatic units connected by polyolefin bridges of varying carbon numbers. These probes exhibit fluorescence excitation and emission patterns similar to many conventional dyes (e.g., fluorescein and tetramethylrhodamine), but they possess enhanced water solubility, photostability, and higher quantum yields. Most cyanide dyes exhibit greater environmental stability than their conventional counterparts, making their fluorescence emission intensity less sensitive to pH and organic encapsulation media (mounting medium). Similar to Alexa Fluors, the excitation wavelengths of Cy-series synthetic dyes can be deliberately tuned for use with common laser and arc discharge sources, and fluorescence emission can be detected using combinations of conventional filters. Cyanide dyes, as reactive dyes or fluorophores, are readily available. Cyanide dyes typically have a broader absorption spectrum than members of the Alexa Fluor family, making them more versatile in the selection of laser excitation sources for confocal microscopy.

[0145] In a preferred embodiment, the detectable element of the compound of the present invention is cyanine dye Cy5.

[0146] In some implementations, the detectable element comprises a benzoindole dye, such as indocyanine green (“ICG”) or indocyanine green residues:

[0147]

[0148] Indocyanine green (ICG) is used in a variety of medical diagnostic applications, such as for monitoring and imaging certain cardiac, hepatic, ophthalmic, and circulatory system diseases. Advantageously, ICG and related compounds exhibit absorption and emission spectra in the near-infrared region. For example, ICG absorbs primarily between 600 nm and 900 nm and emits primarily between 750 nm and 950 nm. Such wavelengths can penetrate biological tissues, enabling imaging of these tissues using ICG and related compounds. Furthermore, the long-standing and widespread use of ICG in medical diagnostic research demonstrates the biocompatibility of these compounds.

[0149] Therefore, in some embodiments, the detectable element comprises a benzoindole dye having the following structure:

[0150]

[0151] Where o is an integer from 1 to 4;

[0152] R2 is a C2-C8 alkyl group, optionally substituted with a sulfonate or carbonate ester;

[0153] Each R3 is independently a C1-C6 alkyl group; and

[0154] L4 is an alkyl linking group with optional substitution, wherein each carbon atom is arbitrarily replaced by a heteroatom.

[0155] More specifically, the benzoindole dye may have the following structure:

[0156]

[0157] For example, Zhang et al. (2005) Chem. Commun. 2005:5887 (DOI:10.1039 / b512315a) describe the synthesis of dyes containing benzoindole. See also U.S. Patent Application Publication No. 2009 / 0214436A1.

[0158] In some embodiments, L4 may be an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced by a heteroatom.

[0159] In some embodiments, it is advantageous to include multiple fluorescent labels, radioactive labels, chelating agents, etc., within the detectable element of the compounds of the present invention. For example, the exemplary compounds labeled “LES12” and “LES13” below include two different fluorescent labels within a single detectable element. As those skilled in the art will understand, such multiple labels can be achieved using conventional coupling chemistry. For example, the fluorescent labels in the compounds “LES12” and “LES13” are coupled using “click” chemistry. An example of a useful intermediate compound containing multiple labels within a detectable element synthesized by “click” chemistry is shown below (“WL938”). This compound contains an azide group, and therefore can readily react with a suitable alkyne-containing reagent in a “click” reaction. As those skilled in the art will understand, the positions of the alkyne and azide groups can also be interchanged if desired.

[0160] The ether-linked leaving element L of the compounds of this invention affects the reactivity of the compounds with the active sites of their target enzymes and can also affect the specificity of targeting specific enzymes. The ether bond in the leaving element of these compounds contrasts with the ester bond in other activity-based probes (such as acyloxymethyl ketones (AOMK)). Compared to ester-linked or other types of probes, ether-linked leaving elements, such as phenolic ether-linked leaving elements, can provide improved in vivo stability.

[0161] In some embodiments, the ether-linked leaving element of the compound of the present invention includes a quencher. The term "quencher" refers to a chemical entity that modulates the luminescence of a fluorophore. In some cases, the quencher itself may be a fluorescent molecule that emits fluorescence at a characteristic wavelength different from that of the fluorescently quenched label. Thus, the fluorophore can act as a quencher when appropriately coupled with other dyes, and vice versa. In these cases, an increase in fluorescence from the acceptor molecule (which has a different wavelength than the donor label) can respectively report the interaction between the labeled compound and its environment (e.g., the active site of the target enzyme). In some cases, the quencher itself does not fluoresce (i.e., the quencher is a "dark acceptor"). Such quenchers include, for example, dabcyl, methyl red, QSY diarylrhodamine dye, etc. In particular, dabcyl (4-dimethylamino-phenylazo)benzoic acid is a common dark quencher widely used in many assays (such as "molecular beacons" for DNA detection) (US Patent No. 5989823). The BHQ series of diazo dyes, known as "black hole quenchers," exhibit a wide absorption range and good luminescence overlap with many fluorophores (PCT International Publication No. WO01 / 86001). The QSY series of dyes from MolecularProbes is another example of dark quencher dyes that have been widely used as quenchers in many bioanalyses (US Patent No. 6399392).

[0162] Specifically, QSY7 is a non-fluorescent diarylrhodamine derivative (US Patent Application Publication No. 2005 / 0014160). QSY21 is a non-fluorescent diarylrhodamine chromophore with strong absorption in the visible spectrum and is an effective fluorescence quencher. The fluorophore / quencher pair is further described in US Patent Application Publication No. 2004 / 0241679.

[0163] IRDye QC-1 (available from LI-COR) is another example of a non-fluorescent dye suitable for use as a quencher for the compounds of this invention. It effectively quenches fluorescence produced by fluorophores over a wide range of wavelengths, from the visible to the near-infrared region.

[0164] In some embodiments of the compounds of the present invention, the leaving group element L is L2-L3-Q, wherein L2 is a phenoxy group, L3 is a linking group, and Q comprises a quencher. The leaving group element may, for example, be:

[0165]

[0166] Each Y group is independently an electron-withdrawing group or a hydrogen group. In such compounds, each Y group can be independently a halogen or a hydrogen group. In a particular compound, the L group (for example) is:

[0167]

[0168] As will be understood by those skilled in the art, the L3 linker group of the departing element described above can be any suitable linker group. In particular, the L3 linker group (for example) can be the L1 group as described above.

[0169] In other specific compounds, the L group (for example) is:

[0170]

[0171] Where R contains the QSY quencher, and n is an integer from 1 to 8. In a particular embodiment, the QSY quencher is a hydrophilic quencher, such as a sulfonated QSY quencher.

[0172] In some specific embodiments, the compounds disclosed herein have the structure of formula (II):

[0173]

[0174] In some implementations, L3-Q is

[0175]

[0176] Where R includes QSY quencher or QC-1 quencher; and

[0177] n is an integer from 1 to 16. More specifically, QSY quenchers are hydrophilic QSY quenchers, such as sulfonated QSY quenchers.

[0178] In some implementations, the QC-1 quencher has the following structure:

[0179]

[0180] In some more specific embodiments, the compounds disclosed herein have the structure of formula (III):

[0181]

[0182] In these implementations, m and n are independently integers from 1 to 16.

[0183] In some implementations, R includes QSY21 or sulfon-QSY21, and D is Cy5.

[0184] Optionally, R contains QC-1 quencher and D contains benzoindole dye.

[0185] In the specific implementation of formula (III), R is

[0186]

[0187] And D is

[0188]

[0189] More specifically, the compound has the following structure:

[0190]

[0191] Other specific non-limiting compound embodiments of the present invention include:

[0192]

[0193] Where R = QSY21 and n = 6;

[0194] R = sulfonyl-QSY21 and n = 6;

[0195] R = QSY21 and n = 2; and

[0196] R = sulfonyl-QSY21 and n = 2.

[0197] Pharmaceutical Composition

[0198] In another aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier. Such compositions are used, for example, for intratissue imaging in animals and also for assessing the activity of enzymes (e.g., proteases) in animals. In particular, for the compounds of the present invention labeled with cathepsins, the pharmaceutical compositions can be used as non-invasive optical imaging tools for cancer cells.

[0199] Pharmaceutically acceptable carriers are known in the art and (e.g.) include aqueous solutions such as water or physiologically buffered saline, or other solvents or excipients (such as ethylene glycol, glycerin, oils (such as olive oil), or injectable organic esters). In a specific embodiment, the aqueous solution is pyrogen-free or substantially pyrogen-free when the pharmaceutical composition is administered to humans. Excipients may be selected (e.g.) to achieve sustained release of the agent or selective targeting of one or more cells, tissues, or organs. The pharmaceutical composition may be in unit-dose form (e.g., tablets, capsules, sprinkle capsules, granules, powders, syrups, suppositories, or injections, etc.). The composition may also be present in transdermal delivery systems (e.g., skin patches).

[0200] Pharmaceutically acceptable carriers may contain physiologically acceptable agents, for example, for stabilizing or increasing the absorption of the compounds of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of pharmaceutically acceptable carrier (including physiologically acceptable agents) depends, for example, on the route of administration of the composition. Pharmaceutical compositions may also include liposomes or other polymer matrices in which, for example, the pharmaceutical compositions of the present invention are already contained. Liposomes, for example, composed of phospholipids or other lipids, are non-toxic, physiologically acceptable, and metabolizable carriers that can be relatively easily manufactured and administered.

[0201] In this article, the phrase “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0202] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or excipient, such as a liquid or solid filler, diluent, additive, solvent, or encapsulating material, relating to the delivery or transport of a target compound from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense of compatibility with other components of the formulation and harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) additives, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn... Oils and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations. See Remington: The Science and Practice of Pharmacy, 20th ed. (edited by Alfonso R. Gennaro), 2000.

[0203] Pharmaceutical compositions comprising the compounds of the present invention may be administered to a subject via any of a variety of routes of administration, including, for example, oral (e.g., as an infiltrator in an aqueous or non-aqueous solution or suspension, tablets, pills, powders, granules, or paste applied to the tongue); sublingual administration; anal, rectal, or vaginal administration (e.g., as a vaginal suppository, cream, or foam); parenteral administration (including intramuscular, intravenous, subcutaneous, or intrathecal administration, e.g., via a sterile solution or suspension); nasal administration; intraperitoneal administration; subcutaneous administration; transdermal administration (e.g., as a patch applied to the skin); or topical administration (e.g., as a cream, ointment, or spray applied to the skin). The compounds may also be formulated for inhalation. In some embodiments, the compounds of the present invention may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for said routes of administration can be found, for example, in the following documents: U.S. Patent Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, and the documents cited in these patent documents.

[0204] Methods of marking and visualization

[0205] In another aspect, the present invention provides a method for visualizing tumors in an animal, the method comprising the step of administering the composition of the present invention to the animal.

[0206] In another aspect, the present invention provides a method for visualizing tumors in an animal, the method comprising administering the composition of the present invention to the animal and measuring a detectable signal generated in the animal due to a reaction between the composition and cysteine ​​cathepsin, wherein the detectable signal is associated with a tumor in the animal.

[0207] In some embodiments of the method, the detectable signal is a fluorescent signal. In some embodiments, the fluorescent signal is generated at the edge of the tumor.

[0208] Administering peptide imaging agents to animals is well known to those skilled in the art. In a preferred embodiment, the agent is administered by injection, but any other suitable method of administration is also considered to be within the scope of this invention.

[0209] The method of the present invention is aimed at labeling and visualizing proteases (particularly cysteine ​​proteases) in animals. Suitable animals include those that express cysteine ​​proteases, particularly those that express cysteine ​​proteases in tumor cells. In a preferred embodiment, the animal is a mammal. In a highly preferred embodiment, the animal is a human. In other preferred embodiments, the animal is livestock or a pet.

[0210] In some embodiments, the method of the present invention includes the step of measuring a detectable signal generated in an animal. Methods for measuring the detectable signal include, but are not limited to, imaging methods, such as fluorescence imaging. In some embodiments, for example, the fluorescence imaging system is the Xenogen IVIS 100 system, but any suitable imaging system can be used.

[0211] It will be apparent to those skilled in the art that other suitable modifications and adjustments can be made to the methods and applications described herein without departing from the scope of the invention or any of its embodiments. While the invention has now been described in detail, it will be more clearly understood by referring to the following examples, which are illustrative only and not intended to limit the scope of the invention.

[0212] Example

[0213] Quenched fluorescent cysteine ​​tissue containing a novel phenoxymethyl ketone (PMK) electrophile Protease imaging probes Synthesis and Characterization

[0214] The aim of this work was to develop a qABP with overall improved in vivo performance compared to existing qABPs for non-invasive optical imaging in cancer. Therefore, it was decided to optimize three key components of the probe: the quencher, the linker group, and the electrophilic "warhead." One of the biggest drawbacks of cysteine ​​cathepsin qABPs reported to date is their poor water solubility. Therefore, a sulfonate group was introduced into the QSY21 quencher (Xing et al., (2005) J. Am. Chem. Soc. 127:4158-9) to improve water solubility, thereby improving the probe's biodistribution. The length of the spacer linking the electrophile and the quencher was also altered to reduce the lipophilicity of the qABP. Finally, novel electrophiles were explored to broaden the range of potential cathepsin targets. Since the expression of some members of the cysteine ​​protease family is upregulated in various cancers (Mohamed & Sloane (2006) Nat. Rev. Cancer (2006) 6:764-75), a brighter fluorescent signal in tumors could be expected if the activity of a broad spectrum of cysteine ​​proteases is probed. To obtain probes with broader reactivity, the size of the electrophile has been reduced, thus increasing reactivity. It has previously been shown that the 2,3,5,6-tetrafluoro-substituted phenoxymethyl ketone (PMK) electrophile is more reactive to cysteine ​​dipeptidyl aminopeptidases than that of 2,6-dimethylbenzoic acid-derived acyloxymethyl ketones (AOMK). Deu et al., (2010) Chem Biol. 17:808-819. The smaller size of PMK also enhances broad reactivity because the binding grooves of some cysteine ​​cathepsins are spatially confined. Blum et al., (2005) Nat. Chem. Biol. 1:203-9; Blum et al., (2007) Nat. Chem. Biol. 3:668-77; Paulick & Bogyo (2011) ACS Chem. Biol. 6:563-72. Furthermore, phenolic ethers are expected to be more stable in vivo compared to AOMK electrophiles (which contain ester bonds that can be degraded by esterases).

[0215] As a starting point for this study, seven analogues (2-8) of qABP GB137(1) were synthesized. Blum et al., (2007) Nat. Chem. Biol. 3:668-77; PCT International Publication No. WO 2014 / 145257. Figure 1A These compounds represent all combinations of two electrophiles, two quenchers, and two linker lengths. All probes were synthesized using a preferred solution-chemistry-based approach as described in the description related to Scheme 1 below. Initially, the specificity and potency of the probes were tested by labeling intact RAW 264.7 cells (a mouse monocytic leukemia macrophage cell line). Figure 1B Several trends in probe properties were observed. All sulfonyl-QSY21-functionalized qABPs (2, 4, 6, and 8) exhibited stronger overall cathepsin labeling performance compared to probes containing the more hydrophobic QSY21 (1, 3, 5, and 7). Interestingly, changes in spacer length resulting from the replacement of the hexyl linker with the ethyl linker did not significantly affect the labeling behavior. Perhaps the most striking finding was that qABPs with PMK electrophiles exhibited a broader cysteine ​​protease labeling profile compared to their AOMK counterparts. Probes 5 through 8 showed robust cathepsin X labeling, and sulfonyl-QSY21-functionalized probes 6 and 8 were able to label higher molecular weight cathepsin L precursor forms. The properties of the fluorescently labeled cathepsins were determined using immunoprecipitation. Figure 4A After titration labeling experiments were performed in live RAW cells, other trends of interest were observed. Figure 1C , 1D ; Figure 4B , 4C The most hydrophobic qABPs (1 and 5) reached their maximum decreasing labeling strength at 0.5 μM, suggesting that their reduced water solubility leads to probe precipitation at higher concentrations. Shorter spacer lengths appear to be beneficial, as all probes carrying ethyl spacers exhibit brighter labeling compared to probes containing hexyl groups. Significant differences in selectivity were observed when compared to AOMKs with PMK. AOMK qABPs preferentially labeled cathepsins S and L, and only labeled cathepsin B at higher concentrations. Surprisingly, AOMK qABPs 2–4 labeled cathepsin X, although previous studies have shown that some other related AOMKs cannot label this target (Paulick & Bogyo (2011) ACS Chem. Biol. 6: 563–72). Even at lower probe concentration levels, PMK qABPs labeled all target cysteine ​​cathepsins with equivalent strength. In summary, these experiments demonstrate that increased hydrophilicity improves labeling strength and that the novel PMK qABP possesses a broader and more complete cysteine ​​cathepsin labeling profile.

[0216] Since PMK qABP 8 is optimized for both overall labeling intensity and spectral cathepsin reactivity, further in vivo studies of this probe were decided upon. To further determine target selectivity, RAW cell lysates were labeled with qABP 8 at progressively increasing concentrations at pH 5.5. These results indicate that the probe is most effective against cathepsins B and X, as labeling was observed at concentrations as low as 5 nM. However, labeling of all cathepsins (B, S, L, X) was saturated with a 500 nM probe concentration. Figure 2AWhen the probe was used for real-time labeling in live RAW cells at a set concentration of 500 nm, rapid saturation of cathepsin X was observed, followed by slower labeling of cathepsin S, L, and B, with the cathepsin B labeling signal progressively increasing, even at 120 minutes. Figure 2B These data suggest that the probe likely enters the cathepsin X pool most rapidly, possibly due to its intracellular or cell surface localization. This also indicates that cathepsin B and X may be other sites within the cell where the probe can enter to varying degrees. To test the stability of the novel PMK probe, the effect of serum exposure on labeling in RAW cells was examined. Figure 1C While initial 4-hour serum pre-exposure to the AOMK probe 1 resulted in nearly 70% loss of target labeling, over 80% of the labeling for PMK qABP 8 was retained. Pretreatment of cells with the cysteine ​​cathepsin inhibitor JPM-OET also blocked over 90% of this labeling. Given the stability and improved labeling properties of the PMK probe, live-cell fluorescence microscopy studies were then performed. These results confirmed that the probe produces a bright and specific labeling signal, and that most of the probe-labeled cathepsins reside in lysosomes. Figure 2D ).

[0217] Given the novel electrophilic positive live-cell labeling properties of PMK, the best-performing PMK qABP 2, 6, and 8 were tested in an orthotopic mouse model of breast cancer. (Tao et al., (2008) BMC Cancer 8:228.) Furthermore, these PMK probes were compared with the initial AOMK probe 1. Figures 3A-3F and Figures 5A-5F 4T1 cells were transplanted into mammary fat pads of Balb / c mice (numbers 2 and 7), and tumor growth was monitored. When tumors formed, the mice were injected intravenously with an equimolar amount of qABP (20 nmol), and Cy5 fluorescence was used for imaging over time in a non-invasive manner. Figure 3A , 3B These results further confirm that qABP 8 is indeed superior. For probe 8, strong tumor-specific fluorescence activation with high overall contrast was clearly observed within the tumor region. This signal continued to increase over time until the end of the time course. Ultimately, probe 8 achieved a 20-fold increase in tumor-specific fluorescence signal compared to probe 1. Probe 6 also showed good tumor-specific contrast, and probe 2 also showed good tumor-specific contrast to a lesser extent, although both still exceeded probe 1 by more than 10 times. Figure 5A , 5BAfter the time course was completed, the tumor was removed and the fluorescence of the ex vivo tumor was measured. The tumor was then homogenized, and the fluorescently labeled proteins were analyzed by SDS-PAGE. Figure 3C and Figure 5C ). In vitro fluorescence and quantification of total cysteine ​​cathepsin labeling showed trends similar to those observed in non-invasive optical imaging studies. Figure 3D and 5D To determine the cellular origin of the probe fluorescence, immunofluorescence staining was performed on tumor tissue sections from probe-labeled mice using the macrophage marker CD68. Figure 3E and Figure 5E Cy5 fluorescence localized to CD68-positive cells; however, not all CD68-positive cells were also positive for probe 8, indicating different activation states of tumor-associated macrophages. More detailed analysis using laser confocal scanning microscopy (CLSM) confirmed that all probe 8-positive cells were CD68-positive, but the probe-labeled cathepsins and CD68 signals did not co-localize to the same vesicles. Figure 3F and Figure 5F In summary, these data confirm that increasing the hydrophilicity of the quencher, shortening the spacer, and introducing a more reactive and less spatially restricted nucleophilic trap result in qABP exhibiting broad cysteine ​​cathepsin reactivity and overall improved in vivo properties.

[0218] Although very different functions have been described for some family members of cysteine ​​proteases (Conus & Simon (2010) Swiss Med. Wkly. 140: w13042), other functions are redundant, and changes in the activity of one cathepsin can affect the activity of other cathepsins. For example, loss of cathepsin B is compensated by increased activity of cathepsin X (Sevenich et al., (2010) Proc. Natl Acad. Sci. USA 107: 2497-502), and upregulation of cathepsin B leads to downregulation of cathepsin L (Gopinathan et al., (2012) Gut 61: 877-84). Therefore, broad-spectrum probes are valuable because they facilitate the reading of multiple cysteine ​​cathepsins in a single assay and allow for comparison of the activities of individual cathepsins. The effectiveness of such broadly reactive ABPs has been demonstrated by broadly reactive serine hydrolase fluorophosphate probes (Liu et al., (1999) Proc. Natl Acad. Sci. USA 96:14694-9) and broadly reactive protease probes MV151 (Verdoes et al., (2006) Chem. Biol. 13:1217-26). Furthermore, due to the high reactivity of PMK-based qABPs to cathepsin X, these backbones can be used to prepare qABPs selective for cysteine ​​cathepsins, which are still poorly understood (Paulick & Bogyo (2011) ACS Chem. Biol. 6:563-72).

[0219] In summary, a novel class of quenching-based fluorescently active probes has been synthesized, which, compared to previously reported AOMK-based probes, incorporate a PMK electrophile with higher reactivity and broader selectivity. The hydrophilicity of qABP is further increased by introducing a sulfonated quencher and shortening the spacer connecting the electrophile and the quencher, resulting in higher water solubility and improved in vivo properties, leading to enhanced contrast in non-invasive optical imaging of cancer.

[0220] method

[0221] Summary

[0222] All resins and reagents were purchased from commercial suppliers and used without further purification. All solvents used were HPLC grade. All water-sensitive reactions were carried out under positive pressure of argon in anhydrous solvents. Reactions were analyzed by LC-MS using an API 150EX single quadrupole mass spectrometer (Applied Biosystems). A C18 column was used. Reversed-phase HPLC was performed using a detector 100 (Amersham Pharmacia Biotech). NMR spectra were recorded on a Varian 400 MHz (400 / 100), Varian 500 MHz (500 / 125), or Varian Inova 600 MHz (600 / 150 MHz) equipped with a pulsed-field gradient attachment. Chemical shifts are shown as ppm (δ) relative to tetramethylsilane as an internal standard. Coupling constants are shown in Hz. The fluorescent gel was scanned using a Typhoon 9400 flatbed laser scanner (GE Healthcare). The labeling intensity within the gel was quantified using ImageJ software. Statistical analysis was performed using Microsoft Excel, and SEM was calculated by dividing sd by the square root of n. Fluorescence microscopy images were acquired on a Zeiss confocal LSM 710 and a Zeiss Axiovert 200M reverse-phase microscope (Carl Zeiss) equipped with 10×, 40×, and 63× objectives. Slidebook software is used to control microscopes and cameras and for data analysis (Intelligent Imaging Innovations).

[0223] qABP synthesis

[0224] The synthetic scheme for synthesizing the following compounds is described in Scheme 1 shown below.

[0225] 2,6-Dimethyl-4–((6-(triphenylmethylamino)hexyl)carbamoyl)benzoic acid (11a). Mono-triphenylmethyl 1,6-hexanediamine acetate (9a) (117.2 mg, 0.28 mmol) was dissolved in DCM, washed with a saturated aqueous solution of NaHCO3, dried over Na2SO4, and concentrated under vacuum. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equivalent), and EDC (54 mg, 0.28 mmol, 1 equivalent) and 2,6-dimethyl-4-benzoic acid (10) (54.4 mg, 0.28 mmol, 1 equivalent) were added. The reaction mixture was stirred overnight and then concentrated under vacuum. The crude product was purified by rapid column chromatography (DCM → DCM solution of 5% MeOH), followed by dissolution in DCM and washing with water, drying over MgSO4 to give 70 mg of product (0.13 mmol, separation yield: 47%).

[0226] 2,6-Dimethyl-4-((2-(triphenylamino)ethyl)carbamoyl)benzoic acid (11b). Mono-triphenylmethylethylenediamine acetate (9b) (97.9 mg, 0.27 mmol) was dissolved in DCM, washed with a saturated aqueous solution of NaHCO3, dried over Na2SO4, and concentrated under vacuum. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1.04 equivalents), and EDC (61 mg, 0.32 mmol, 1.2 equivalents) and 2,6-dimethyl-terebenzoic acid (10) (52 mg, 0.27 mmol, 1 equivalent) were added. The reaction mixture was stirred overnight and then concentrated under vacuum. The crude product was purified by rapid column chromatography (DCM → DCM solution of 5% MeOH), followed by dissolution in DCM and washing with water, drying over MgSO4 to give 28 mg of product (0.06 mmol, separation yield: 22%).

[0227] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(6-(triphenylmethylamino)hexyl)benzamide (13a). Mono-triphenylmethyl 1,6-hexanediamine acetate (9a) (117.2 mg, 0.28 mmol) was dissolved in DCM, washed with a saturated aqueous solution of NaHCO3, dried over Na2SO4, and concentrated under vacuum. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equivalent), and EDC (54 mg, 0.28 mmol, 1 equivalent) and 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (12) (59 mg, 0.28 mmol, 1 equivalent) were added. The reaction mixture was stirred overnight and then concentrated under vacuum. The crude product was purified by rapid column chromatography (15% -> 30% ethyl acetate in hexane) to give 90 mg of product (0.16 mmol, separation yield: 58%).

[0228] 2,3,5,6-Tetrafluoro-4-hydroxy-N-(2-(triphenylmethylamino)ethyl)benzamide (13b). Mono-triphenylmethylethylenediamine acetate (9b) (100 mg, 0.28 mmol) was dissolved in DCM, washed with a saturated aqueous solution of NaHCO3, dried over Na2SO4, and concentrated under vacuum. The amine was dissolved in DMF and HOBt monohydrate (43 mg, 0.28 mmol, 1 equivalent), and EDC (54 mg, 0.28 mmol, 1 equivalent) and 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (12) (59 mg, 0.28 mmol, 1 equivalent) were added. The reaction mixture was stirred overnight and then concentrated under vacuum. The crude product was purified by rapid column chromatography (20% -> 35% ethyl acetate in hexane) to give 90 mg of product (0.18 mmol, separation yield: 65%). 1H NMR (400MHz, DMSO) δ=8.77(t,J=6.0,1H),7.39(d,J=7.8,6H),7.27(t,J=7.7,6H ),7.17(t,J=7.2,3H),3.40–3.35(m,2H),2.86–2.77(m,1H),2.14–2.04(m,2H).

[0229]

[0230] Scheme 1. Reagents and conditions: i. EDC, HOBt, DMF. ii. a) KF, DMF. b) 1% TFA, DCM. iii. a) QSY21-NHS or sulfonated QSY21-NHS, DiPEA, DMSO. b) TFA / DCM = 1 / 1. c) Cy5-NHS, DiPEA, DMSO. iv. a) KF, DMF, 80℃. b) 1% TFA, DCM.

[0231] Intermediate 15. Potassium fluoride (3 mg, 52 μmol, 3 equivalents) was suspended in DMF by sonication for 5 minutes, followed by the addition of carboxylic acid 11a (10 mg, 19 μmol, 1.1 equivalents). The reaction mixture was stirred for 10 minutes, followed by the addition of chloromethyl ketone 14 (9.7 mg, 17.3 μmol, 1 equivalent). After 2 hours, the reaction mixture was concentrated under vacuum, and the crude product was dissolved in a 1% TFA solution in DCM and stirred for 30 minutes, followed by quenching with triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), the product was analyzed by HPLC (preparative reversed-phase C). 18 The title compound was purified by column chromatography (CH3CN / H2O 0.1% TFA, 15:85 to 55:45 over 20 min; 5 mL / min), followed by lyophilization to give a white powder 15 (3.12 mg, 3.46 μmol, 20% yield after two steps).

[0232] Intermediate 16. Potassium fluoride (3 mg, 52 μmol, 3 equivalents) was suspended in DMF by sonication for 5 min, followed by the addition of carboxylic acid 11b (9.5 mg, 20 μmol, 1.1 equivalents). The reaction mixture was stirred for 10 min, followed by the addition of chloromethyl ketone 14 (10 mg, 17.9 μmol, 1 equivalent). After 1.5 hours, the reaction mixture was concentrated under vacuum, and the crude product was dissolved in a 1% TFA solution in DCM and stirred for 30 min, followed by quenching with triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), the product was analyzed by HPLC (preparative reversed-phase C). 18The intermediate 16 was purified by column chromatography (CH3CN / H2O 0.1% TFA, 15:85 to 55:45 over 20 min; 5 mL / min), followed by lyophilization to give a white powder (3.99 mg, 4.57 μmol, yield 26% after two steps). 1 H NMR(500MHz,CD3OD)δ7.80(s,1H),7.42(s,1H),7.35–7.18(m,10H),5.06(s,2H),4.85–4.78(m, 2H),4.42(dd,J=13.1,6.2Hz,1H),4.37(dd,J=10.1,4.0Hz,1H),3.64(t,J=5.7Hz,2H),3.18(t,J =4.8Hz,2H),3.12(dd,J=13.7,7.0Hz,1H),3.01(t,J=7.3Hz,2H),2.94(dd,J=13.6,8.9Hz,1H), 2.41(s,3H),2.34(s,3H),1.92–1.82(m,1H),1.67–1.57(m,1H),1.49–1.26(m,4H),1.42(s,9H).

[0233] Intermediate 17. Potassium fluoride (6.3 mg, 108 μmol, 3 equivalents) was suspended in DMF by sonication for 5 min, followed by the addition of phenol 13a (21.5 mg, 39 μmol, 1.1 equivalents). The reaction mixture was stirred for 10 min, followed by the addition of chloromethyl ketone 14 (20 mg, 36 μmol, 1 equivalent). The reaction mixture was stirred at 80 °C for 5 h, followed by vacuum concentration. The crude product was dissolved in a 1% TFA solution in DCM and stirred for 30 min, followed by quenching with triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), the product was analyzed by HPLC (preparative reversed-phase C). 18 The compound was purified by column chromatography (CH3CN / H2O 0.1% TFA, 25:75 to 70:30 for 20 min; 5 mL / min), followed by lyophilization to give the title compound as a white powder (16.6 mg, 17.5 μmol, 49% yield after two steps). 1H NMR (500MHz, CD3OD) δ7.29(m,10H),5.07(s,2H),4.86(m,2H),4.44(m,2H),3.41(t,J=6.8,2H),3.10(dd,J=13.5,7.0,1H),3.02(t,J=6 .8,2H),2.97–2.91(m,3H),1.93–1.81(m,1H),1.73–1.62(m,4H),1.62–1.53(m,1H),1.51–1.46(m,4H),1.43(s,9H),1.45–1.25(m,4H).

[0234] Intermediate 18. Potassium fluoride (6.3 mg, 108 μmol, 3 equivalents) was suspended in DMF by sonication for 5 min, followed by the addition of phenol 13b (19.4 mg, 39 μmol, 1.1 equivalents). The reaction mixture was stirred for 10 min, followed by the addition of chloromethyl ketone 14 (20 mg, 36 μmol, 1 equivalent). The reaction mixture was stirred at 80 °C for 3 h, followed by vacuum concentration. The crude product was dissolved in a 1% TFA solution in DCM and stirred for 30 min, followed by quenching with triisopropylsilane until the solution became colorless. After co-evaporation with toluene (3x), the product was analyzed by HPLC (preparative reversed-phase C). 18 The compound was purified by column chromatography (CH3CN / H2O 0.1% TFA, 20:80 to 60:40 for 20 min; 5 mL / min) followed by lyophilization to give the title compound as a white powder (15.4 mg, 17.3 μmol, 48% yield after two steps). 1 H NMR (400MHz, CD3OD) δ=7.36–7.12(m,10H),5.05(s,2H),4.86–4.81(m,2H),4.42–4.37(m,2H),3.64(t,J=6.5,2H),3.14(t,J=6.5,2H),3.08 (dd,J=13.9,7.2,1H),2.99(t,J=6.5,2H),2.91(dd,J=13.9,8.4,1H),1.90–1.78(m,1H),1.62–1.48(m,1H),1.41(s,9H),1.46–1.20(m,4H).

[0235] Probe 1 (GB137). Intermediate 15 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.39 mg, 1.7 μmol, 1 equivalent) and DiPEA (1.5 μl, 8.5 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18The QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 40:60 to 80:20 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 2.42 mg of the corresponding TFA salt (1.6 μmol, 95% yield after two steps). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (1.3 mg, 1.76 μmol, 1.1 equivalent) and DiPEA (1.4 μl, 8 μmol, 5 equivalent) were added. After 1 hour, the amide was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at 40:60 to 75:25 for 20 minutes (5 mL / min), and then lyophilized to obtain probe 1 (2.0 mg, 0.99 μmol, 62%) as a dark blue powder.

[0236] Probe 2 (BMV122). Intermediate 15 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and sulfonyl-QSY21-NHS (1.66 mg, 1.7 μmol, 1 equivalent) and DiPEA (1.5 μl, 8.5 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18 The sulfonyl-QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 30:70 to 70:30 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 2.29 mg of the corresponding TFA salt (1.39 μmol, 81% yield after two steps). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (1.1 mg, 1.5 μmol, 1.1 equivalent) and DiPEA (1.2 μl, 7 μmol, 5 equivalent) were added. After 1 hour, the amine was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at 15:85 to 50:50 for 20 minutes (5 mL / min), and then lyophilized to obtain a dark blue powder of probe 2 (1.83 mg, 0.84 μmol, 61%).

[0237] Probe 3 (BMV145). Intermediate 16 (1.5 mg, 1.7 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.39 mg, 1.7 μmol, 1 equivalent) and DiPEA (1.5 μl, 8.5 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18 The QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 40:60 to 80:20 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 0.86 mg of the corresponding TFA salt (0.6 μmol, 35% yield after two-step separation). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (0.5 mg, 0.66 μmol, 1.1 equivalent) and DiPEA (0.57 μl, 3.3 μmol, 5 equivalent) were added. After 1 hour, the amide was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at a rate of 40:60 to 75:25 for 20 minutes (5 mL / min), and then lyophilized to obtain a dark blue powder, probe 3 (0.67 mg, 0.34 μmol, 57%).

[0238] Probe 4 (BMV146). Intermediate 16 (1.0 mg, 1.2 μmol) was dissolved in DMSO (50 μl), and sulfonyl-QSY21-NHS (1.25 mg, 1.2 μmol, 1 equivalent) and DiPEA (1.05 μl, 6 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18 The sulfonyl-QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 20:80 to 80:20 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 1.06 mg of the corresponding TFA salt (0.66 μmol, 55% yield after two steps). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (0.55 mg, 0.73 μmol, 1.1 equivalent) and DiPEA (0.64 μl, 3.65 μmol, 5 equivalent) were added. After 1 hour, the amine was purified by HPLC (preparative reversed-phase C20). 18The column was filled with CH3CN / H2O 0.1% TFA at 15:85 to 50:50 for 20 minutes (5 mL / min), and then lyophilized to obtain a dark blue powder, probe 4 (0.63 mg, 0.3 μmol, 45%).

[0239] Probe 5 (BMV118). Intermediate 17 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.0 mg, 1.3 μmol, 1 equivalent) and DiPEA (1.13 μl, 6.5 μmol, 5 equivalents) were added. After 2 hours, the sample was analyzed by HPLC (preparative reversed-phase C). 18 The QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 40:60 to 80:20 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 2.0 mg of the corresponding TFA salt (1.3 μmol, quantified after two steps). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (1.0 mg, 1.3 μmol, 1 equivalent) and DiPEA (1.1 μl, 6.5 μmol, 5 equivalent) were added. After 1 hour, it was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at a rate of 40:60 to 85:15 for 20 minutes (5 mL / min), and then lyophilized to obtain a dark blue powder of probe 5 (1.91 mg, 0.94 μmol, 72%).

[0240] Probe 6 (BMV119). Intermediate 17 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and sulfonyl-QSY21-NHS (1.35 mg, 1.3 μmol, 1 equivalent) and DiPEA (1.13 μl, 6.5 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18The sulfonyl-QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 30:70 to 90:10 for 20 min; 5 mL / min) and then lyophilized. To remove the Boc protecting group, the resulting dark blue powder was dissolved in TFA / DCM (1 / 1) and reacted for 30 min, followed by co-evaporation with toluene (3x) to give 1.98 mg of the corresponding TFA salt (0.9 μmol, 70% yield after two steps). The amine was dissolved in DMSO (50 μl) and Cy5-NHS (0.7 mg, 0.9 μmol, 1.1 equivalence) and DiPEA (0.8 μl, 4.5 μmol, 5 equivalence) were added. After 1 hour, the amine was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at 15:85 to 50:50 for 20 minutes (5 mL / min), and then lyophilized to obtain a dark blue powder, probe 6 (1.63 mg, 0.74 μmol, 82%).

[0241] Probe 7 (BMV108). Intermediate 18 (1.2 mg, 1.3 μmol) was dissolved in DMSO (50 μl), and QSY21-NHS (1.2 mg, 1.4 μmol, 1.1 equivalents) and DiPEA (1.13 μl, 6.5 μmol, 5 equivalents) were added. After 1 hour, the sample was analyzed by HPLC (preparative reversed-phase C). 18 The QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 30:70 to 70:30 for 20 min; 5 mL / min) and then lyophilized to give a deep blue powder (1.43 mg, 0.99 μmol, 76%). It was then reacted in TFA / DCM (1 / 1) for 30 min to remove the Boc protecting group, followed by co-evaporation with toluene (3x). The TFA salt was dissolved in DMSO (50 μl) and Cy5-NHS (0.83 mg, 1.1 μmol, 1.1 equivalence) and DiPEA (0.88 μl, 5 μmol, 5 equivalence) were added. After 1 hour, it was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at a rate of 30:70 to 70:30 for 20 minutes (5 mL / min), followed by lyophilization to obtain a dark blue powder of probe 7 (0.95 mg, 0.48 μmol, yield 49% after two steps).

[0242] Probe 8 (BMV109). Intermediate 18 (5.8 mg, 6.5 μmol) was dissolved in DMSO (100 μl), and sulfonyl-QSY21-NHS (9.75 mg, 10.39 μmol, 1.6 equivalents) and DiPEA (8.4 μl, 50.5 μmol, 7.8 equivalents) were added. The mixture was stirred overnight. The sample was analyzed by HPLC (preparative reversed-phase C2000). 18 The sulfonyl-QSY21 amide was purified by HPLC (CH3CN / H2O 0.1% TFA, 25:75 to 55:45 for 20 min; 5 mL / min) and then lyophilized to obtain a deep blue powder. The Boc protecting group was then removed by reaction in TFA / DCM (1 / 1) for 30 min, followed by co-evaporation with toluene (3x). The residue was dissolved in DMSO (250 μl) and Cy5-NHS (10.5 mg, 13.9 μmol, 2.1 equivalences) and DiPEA (12 μl, 72 μmol, 11 equivalences) were added. After 4 hours, the amide was purified by HPLC (preparative reversed-phase C20). 18 The column was filled with CH3CN / H2O 0.1% TFA at 25:75 to 45:55 for 20 minutes (5 mL / min), followed by lyophilization to obtain a dark blue powder of probe 8 (7.74 mg, 4.61 μmol, yield after three steps: 71%). 1H NMR (600MHz, CD3CN) δ8.12–8.08(m,1H),8.01–7.93(m,2H),7.89–7.85(m,2H),7.75(dd,J=12.0,1.5H z,2H),7.72(dd,J=8.4,1.7Hz,1H),7.69(dd,J=8.3,1.2Hz,1H),7.66(s,2H),7.62–7.57(m,2H),7.51 (dd,J=8.4,5.1Hz,2H),7.46(d,J=9.4Hz,2H),7.41–7.35(m,3H),7.24(s,1H),7.22(s,1H),7.21–7.1 4(m,6H),7.13–7.09(m,6H),7.05(dd,J=8.8,4.6Hz,1H),6.39(t,J=12.8Hz,1H),6.11(t,J=12.6Hz,1 H),4.87(q,J=12.7Hz,2H),4.83(dd,J=39.7,14.1Hz,2H),4.23–4.12(m,4H),3.93(q,J=7.2Hz,2H),3 .86(t,J=7.4Hz,2H),3.34(dd,J=6.7,4.1Hz,2H),3.28–3.15(m,9H),3.04–2.92(m,3H),2.80–2.74(m ,1H),2.45(t,J=11.9Hz,2H),2.15–2.09(m,1H),2.09–2.03(m,2H),1.74–1.58(m,7H),1.57(s,6H),1 .55(s,6H),1.49(dd,J=15.1,7.4Hz,4H),1.35–1.22(m,7H),1.20(t,J=7.3Hz,3H),1.16–1.12(m,4H).

[0243] Cell culture and labeling of live cells and cell lysates

[0244] RAW cells were cultured in DMEM (GIBCO) medium supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). 4T1 cells (ATCC) were cultured in RPMI (GIBCO) medium supplemented with 10% fetal bovine serum (GIBCO FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). All cells were cultured at 37°C in a humidified incubator with 5% CO2. Unless otherwise specified, for intact cell labeling, cells were exposed to the probe (in DMSO, 500x) in the medium and incubated at 37°C for 2 hours. Designated cells were pre-cultured with the inhibitor JPM-OET (in DMSO, 500x) for 1 hour or exposed to mouse serum (1 μl of probe in DMSO stock solution added to 9 μl of serum) 4 hours prior to cell addition. After labeling, cells were washed with PBS and resuspended in hypotonic lysis buffer (50 mM PIPES (pH 7.4), 10 mM potassium chloride, 5 mM magnesium chloride, 2 mM EDTA, 4 mM DTT, and 1% NP-40), placed on ice for 15 min, centrifuged at 4°C for 30 min, and the supernatant was collected. Protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was denatured by adding 4×SDS sample buffer and heating at 100°C for 3 min, analyzed by SDS-PAGE (15%), and the labeled proteases were visualized by scanning the gel with a Typhoon imager (GE Healthcare). Labeling intensity was quantified using ImageJ software. For labeling cathepsins in cell lysates, cells were collected, washed with PBS, and resuspended in citrate buffer (50 mM citrate buffer (pH 5.5), 5 mM DDT, 0.5% CHAPS, and 0.1% Triton X). After placing on ice for 15 minutes and centrifuging at 4°C for 30 minutes, the supernatant was collected, and protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was exposed to the specified probe (200x in DMSO) for 1 hour at 37°C. 4×SDS sample buffer was added, and the protein was denatured at 100°C for 3 minutes, followed by analysis as described above. For live-cell microscopy, RAW cells were centrifuged at 1.10... 5Cells were seeded at a density in phenol red-free complete medium (in vitro system) in 35 mm glass dishes and cultured overnight. Cells were then exposed to either DMSO or 1 μM probe (500x in DMSO) for 2 hours. For the final hour, green lysosomal tracer (final concentration 200 nM, 1000x in DMSO) was added to the cells. Selected cells were pre-cultured for 1 hour with the inhibitor JPM-OET (500x in DMSO). Cells were imaged at 40x magnification using a Zeiss Axiovert 200 μM confocal microscope in both Cy5 and FITC channels.

[0245] animal models

[0246] All animal care and testing were conducted in accordance with current guidelines from the National Institutes of Health and the Stanford University Institutional Animal Care and Use Committee. Female BALB / c mice (6–8 weeks old, Jackson Laboratory) were injected with 1.10% PBS into fat pads numbered 2 and 7 under isoflurane anesthesia. 5 4T1 cells (ATCC) were used to monitor tumor growth. Hair in the target area was removed 24 hours prior to imaging using Nair Lotion. On day 10, 100 μL of the designated probe (20 nmol; 0.8 nmol g) was administered via tail vein. -1Following injection, mice were non-invasively imaged at specified time points using the IVIS 100 system (Xenogen). Images were analyzed using Living Image software (PerkinElmer). After the final time point, mice were anesthetized with isoflurane and euthanized by cervical dislocation. For in vitro fluorescence measurements and evaluation of in vivo probe labeling, tumors were removed, and imaging was performed using an FMT 2500 (PerkinElmer). Tissues were sonicated (on ice for 1 minute) in citrate buffer (50 mM citrate buffer (pH 5.5), 5 mM DDT, 0.5% CHAPS, 0.1% Triton X). After centrifugation at 4°C for 30 minutes, the supernatant was collected, and protein concentration was determined using a BCA kit (Pierce). 40 μg of total protein was denatured in SDS sample buffer at 100°C for 3 minutes and analyzed as described above. For immunofluorescence, excised tumors were cultured in PBS with 4% PFA for 6 hours at 4°C, followed by overnight culture in 30% sucrose solution and tissue freezing in OCT medium. 6 μm sections were fixed in acetone, blocked with PNB blocking buffer, and co-cultured overnight with rat anti-mouse CD68 (1:1000; Serotec). Goat anti-rat conjugated with Alexa Fluor-488 (1:500; Invitrogen) was cultured at room temperature for 1 hour. Sections were then stained with DAPI (2 μg / mL; Invitrogen) for 5 minutes and encapsulated in ProLong Gold encapsulation medium (Invitrogen). The tissues were then visualized using a Zeiss Axiovert 200M microscope.

[0247] Synthesis and characterization of indocyanine green labeled imaging probes

[0248] The following scheme illustrates the synthesis of an imaging probe comprising an indocyanine green detectable element and a QC-1 quencher:

[0249]

[0250] The Boc-protected peptide used in the second step was prepared as described above. The product of the coupling reaction with QC-1 and ICG was confirmed by liquid chromatography-mass spectrometry (“LCMS”).

[0251] In in vivo and in vitro studies, probes containing the ICG fluorophore and QC-1 quencher (BMV109-ICG) were compared with probes containing the Dylight780 fluorophore and QC-1 quencher (BMV109-Dylight780). Figure 6A and 6BAs shown, compared with Dylight780-labeled probes, ICG-labeled probes exhibited improved tumor uptake and lower background signal (see in particular). Figure 6B (The case of using a 50 nmol dose).

[0252]

[0253] All patents, patent publications and other publicly available documents mentioned in this article are incorporated herein by reference as if each reference had been independently and specifically incorporated herein.

[0254] Although specific embodiments have been provided, the foregoing description is illustrative and not restrictive. Any one or more features of the foregoing embodiments can be combined in any manner with one or more features of any other embodiment of the invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon reading this specification. Therefore, the scope of the invention should be determined by reference to the appended claims and their equivalents.

Claims

1. A compound for labeling proteases, having the following formula (II): Where D contains benzoindole dyes substituted with sulfonyl groups or carbonates; L1 is a linking group; AA1 is an amino acid side chain; U is O, NH, or S; R1 is an alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl or protecting group, and is optionally substituted by one to three A groups; Each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, arylalkoxy, arylanoyl, arylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylanoyl, heteroarylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloanoyl, cycloamino, heterocyclic, heterocyclicoxy, heterocyclicamino, heterocyclic alkyl, heterocyclic alkoxy, heterocyclic alkanoyl, heterocyclic alkylamino, hydroxyl, thiol, amino, alkanoylamino, arylanoylamino, alkylcarboxyl, carbonate, carbamate, guanidinyl, ureyl, halogen, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfamino, or azide; L3 is a linking group; and Q contains a quenching agent.

2. The compound according to claim 1, wherein the benzoindole dye has the following structure:

3. The compound according to claim 1, wherein the benzoindole dye has the following structure:

4. The compound according to claim 1, wherein L1 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced by a heteroatom.

5. The compound according to claim 1, wherein AA1 is an aralkyl amino acid side chain, optionally substituted with one to three A groups.

6. The compound according to claim 1, wherein U is O.

7. The compound according to claim 1, wherein L3 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced by a heteroatom.

8. The compound according to claim 1, wherein L3-Q is Where R includes QSY quencher or QC-1 quencher; and n is an integer from 1 to 8.

9. The compound according to claim 8, wherein the QSY quencher is a hydrophilic QSY quencher.

10. The compound according to claim 9, wherein the hydrophilic QSY quencher is a sulfonyl-QSY quencher.

11. The compound according to claim 8, wherein the QC-1 quencher has the following structure:

12. The compound according to claim 1, having formula (III): Where R includes either QSY quencher or QC-1 quencher; and m and n are independent integers from 1 to 8.

13. The compound according to claim 12, wherein R is: And D is:

14. A compound having the following structure:

15. The compound according to claim 14, wherein the compound has a structure according to the following formula:

16. A composition for labeling proteases in animals, comprising the compound of any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. A method for labeling proteases in an animal, comprising the step of administering the composition of claim 16 to the animal.

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